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<p>Myocardial ischemia-reperfusion (I/R) injury is a complex condition characterized by oxidative stress, inflammation, and mitochondrial dysfunction. Ferroptosis, an iron-dependent form of regulated cell death, plays a critical role in cardiomyocyte damage during I/R. Inhibiting ferroptosis has been shown to reduce myocardial injury and improve cardiac function, making it a promising therapeutic target for enhancing clinical outcomes. An in vivo I/R model was established, and infarct size was assessed using triphenyltetrazolium chloride (TTC) staining. Histological changes were analyzed using hematoxylin and eosin (H&amp;E) staining, Masson, immunohistochemistry (IHC), and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. Cellular experiments included analyses of reactive oxygen species (ROS), lipid peroxidation, and iron content using specific fluorescent probes, as well as enzymatic markers measured with commercial assay kits. Cell viability and senescence were evaluated using the Cell Counting Kit-8 (CCK-8) assay and senescence-associated β-galactosidase (SA–β-gal) staining, respectively. Mitochondrial ultrastructure was examined using transmission electron microscopy (TEM), while molecular mechanisms, including DNA methylation, were investigated using methylation-specific polymerase chain reaction (MSP). Interactions among pleckstrin homology-like domain family A member 3 (PHLDA3), DNA (cytosine-5)-methyltransferase 3&#xa0;A (DNMT3a), and polypyrimidine tract binding protein 1 (PTBP1) were evaluated using chromatin immunoprecipitation (ChIP) and RNA pull-down assays. Silencing of PHLDA3 demonstrated protective effects against I/R injury by attenuating cardiomyocyte ferroptosis and cardiac microvascular endothelial cells (CMECs) senescence under hypoxia/reoxygenation (H/R) conditions in vitro and I/R injury in vivo. Mechanistically, downregulated DNMT3a was responsible for PHLDA3 hypomethylation. Furthermore, PTBP1 was identified as an upstream RNA-binding protein that destabilized DNMT3a mRNA during H/R injury, indirectly enhancing PHLDA3 expression. Our findings suggest that PTBP1-mediated DNMT3a downregulation contributes to PHLDA3 hypomethylation, which may promote cardiomyocyte ferroptosis and subsequent microvascular endothelial cell senescence during I/R injury.</p>

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PHLDA3 hypomethylation at the mercy of PTBP1-mediated DNMT3a decay prompts ferroptosis of cardiomyocytes to accelerate microvascular endothelial cell senescence following ischemia/reperfusion injury

  • 期刊论文

  • 发表日期：

    2026年7月22日

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  • Xiaoxiao Hou,
  • Ying Chen,
  • Qixun Xu,
  • Jiaxin Zhong,
  • Yudan Long,
  • Kai Liu

摘要

Myocardial ischemia-reperfusion (I/R) injury is a complex condition characterized by oxidative stress, inflammation, and mitochondrial dysfunction. Ferroptosis, an iron-dependent form of regulated cell death, plays a critical role in cardiomyocyte damage during I/R. Inhibiting ferroptosis has been shown to reduce myocardial injury and improve cardiac function, making it a promising therapeutic target for enhancing clinical outcomes. An in vivo I/R model was established, and infarct size was assessed using triphenyltetrazolium chloride (TTC) staining. Histological changes were analyzed using hematoxylin and eosin (H&E) staining, Masson, immunohistochemistry (IHC), and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. Cellular experiments included analyses of reactive oxygen species (ROS), lipid peroxidation, and iron content using specific fluorescent probes, as well as enzymatic markers measured with commercial assay kits. Cell viability and senescence were evaluated using the Cell Counting Kit-8 (CCK-8) assay and senescence-associated β-galactosidase (SA–β-gal) staining, respectively. Mitochondrial ultrastructure was examined using transmission electron microscopy (TEM), while molecular mechanisms, including DNA methylation, were investigated using methylation-specific polymerase chain reaction (MSP). Interactions among pleckstrin homology-like domain family A member 3 (PHLDA3), DNA (cytosine-5)-methyltransferase 3 A (DNMT3a), and polypyrimidine tract binding protein 1 (PTBP1) were evaluated using chromatin immunoprecipitation (ChIP) and RNA pull-down assays. Silencing of PHLDA3 demonstrated protective effects against I/R injury by attenuating cardiomyocyte ferroptosis and cardiac microvascular endothelial cells (CMECs) senescence under hypoxia/reoxygenation (H/R) conditions in vitro and I/R injury in vivo. Mechanistically, downregulated DNMT3a was responsible for PHLDA3 hypomethylation. Furthermore, PTBP1 was identified as an upstream RNA-binding protein that destabilized DNMT3a mRNA during H/R injury, indirectly enhancing PHLDA3 expression. Our findings suggest that PTBP1-mediated DNMT3a downregulation contributes to PHLDA3 hypomethylation, which may promote cardiomyocyte ferroptosis and subsequent microvascular endothelial cell senescence during I/R injury.